Alterations in the cellular genes which directly or indirectly control cellular growth (proliferation) and differentiation are considered to be causative events leading to the development of tumors and cancers (see, generally, Weinberg, Sci. American 275:62, 1996). There are many families of genes presently implicated in human tumor formation. Members of one such family, the ras gene family, are frequently found to be mutated in human tumors. In their normal state, proteins produced by the ras genes are thought to be involved in normal cell growth and maturation. Mutation of the ras gene, causing an amino acid alteration at one of three critical positions in the protein product, results in conversion to a form which is implicated in tumor formation. A gene having such a mutation is said to be "activated." It is thought that such a point mutation leading to ras activation can be induced by carcinogens or other environmental factors. Over 90% of pancreatic adenocarcinomas, about 50% of adenomas and adenocarcinomas of the colon, about 50% of adenocarcinomas of the lung and carcinomas of the thyroid, and a large fraction of malignancies of the blood such as acute myeloid leukemia and myelodysplastic syndrome have been found to contain activated ras genes. Overall, some 10 to 20% of human tumors have a mutation in one of the three ras genes (H-ras, K-ras and N-ras).
It is presently believed that inhibiting expression of activated cancer-associated genes in a particular tumor cell might force the cell back into a more normal growth habit. For example, Feramisco et al. (Nature, 314:639, 1985) demonstrated that cells transformed to a malignant state with an activated ras gene slow their rate of proliferation and adopt a more normal appearance when microinjected with an antibody which binds to the protein product of the ras gene. This has been interpreted as support for the involvement of the product of the activated ras gene in the uncontrolled growth typical of cancer cells.
The H-ras gene has recently been implicated in a serious cardiac arrhythmia called long Q-T syndrome, a hereditary condition which often causes sudden death if treatment is not given immediately. Frequently there are no symptoms prior to the onset of the erratic heartbeat. Whether the H-ras gene is precisely responsible for long Q-T syndrome is unclear. However, there is an extremely high correlation between inheritance of this syndrome and the presence of a particular variant of the chromosome 11 region surrounding the H-ras gene. Therefore, the H-ras gene is a useful indicator of increased risk of sudden cardiac death due to the long Q-T syndrome.
There is a great desire to provide compositions of matter which can modulate the expression of the ras gene, and particularly to provide compositions of matter which specifically modulate the expression of the activated form of the ras gene. Inhibition of K-ras gene expression has been accomplished using retroviral vectors or plasmid vectors which express a 2-kilobase segment of the K-ras gene RNA in antisense orientation (Mukhopadhyay et al., Cancer Research 51:1744, 1991; PCT Patent Application PCT/US92/01852 (WO 92/15680). Georges et al., Cancer Research, 53:1743, 1993).
Antisense oligonucleotide inhibition of expression has proven to be a useful tool in understanding the role(s) of various cancer-associated gene families. Antisense oligonucleotides are small oligonucleotides which are complementary to the "sense" (coding strand) of a given gene, and are thus also complementary to, and thus able to stably and specifically hybridize with, the mRNA transcript of the gene. Holt et al. (Mol. Cell Biol. 8:963, 1988) state that antisense oligonucleotides designed to hybridize specifically with (i.e., "targeted to") mRNA transcripts of the c-myc gene inhibit proliferation and induce differentiation when added to cultured HL60 leukemic cells. Anfossi et al. (Proc. Natl Acad. Sci. 86:3379, 1989) state that antisense oligonucleotides targeted to the c-myb gene inhibit proliferation of human myeloid leukemia cell lines. Wickstrom et al. (Proc. Nat. Acad. Sci. 85:1028, 1988) state that expression of the protein product of the c-myc gene and proliferation of HL60 cultured leukemic cells are both inhibited by antisense oligonucleotides hybridizing specifically with c-myc mRNA.
With specific regard to oligonucleotides having ras sequences, U.S. Pat. No. 4,871,838 to Bos et al. discloses oligonucleotides complementary to a mutation in codon 13 of N-ras to detect this mutation. Helene and co-workers have reported the selective inhibition of activated (codon 12 G.fwdarw.T transition) H-ras mRNA expression using a 9-mer phosphodiester linked to an acridine intercalating agent and/or a hydrophobic tail; this compound displayed selective targeting of mutant ras message in both Rnase H and cell proliferation assays at low micromolar concentrations (Saison-Behmoaras et al., EMBO J. 10:1111, 1991). Chang et al. (Biochemistry 30:8283, 1991) disclose selective targeting of a mutant H-ras message, specifically, H-ras codon 61 containing an A.fwdarw.T transversion, with an 11-mer methylphosphonate oligonucleotide or its psoralen derivative. These compounds, which required concentrations of 7.5-150 .mu.M for activity, were shown by immunoprecipitation to selectively inhibit mutant p21.sup.H-ras expression relative to wildtype p21.sup.H-ras.
Although it has been recognized that antisense oligonucleotides have great therapeutic potential, there remains a long-felt need for pharmaceutical compositions and methods that could positively alter the in vivo stability, concentration, and distribution of such oligonucleotides. Enhanced biostability of antisense oligonucleotides in a mammal would generally be preferred for improved delivery of the oligonucleotide to its intended target tissue(s) with potentially less frequent dosing. For antisense oligonucleotides targeted to oncogenic molecules, enhanced distribution to tumor tissues would be preferred.